Respiratory electron transport complexes are organized as individual entities or combined as large supercomplexes (SC). Gram-negative bacteria deploy a mitochondrial-like cytochrome (cyt) bc1 (Complex III, CIII2), and may have specific cbb3-type cyt c oxidases (Complex IV, CIV) instead of the canonical aa3-type CIV. Electron transfer between these complexes is mediated by soluble (c2) and membrane-anchored (cy) cyts. Here, we report the structure of an engineered bc1-cbb3 type SC (CIII2CIV, 5.2 Å resolution) and three conformers of native CIII2 (3.3 Å resolution). The SC is active in vivo and in vitro, contains all catalytic subunits and cofactors, and two extra transmembrane helices attributed to cyt cy and the assembly factor CcoH. The cyt cy is integral to SC, its cyt domain is mobile and it conveys electrons to CIV differently than cyt c2. The successful production of a native-like functional SC and determination of its structure illustrate the characteristics of membrane-confined and membrane-external respiratory electron transport pathways in Gram-negative bacteria.
Bibliographical noteFunding Information:
This work was supported partly by the NIH grants GM 38237 to F.D., GM123233 to K.M., GM110174 and AI118891 to B.A.G., T32-GM008275 to T.V., T32-GM071339 to H.J.K., and partly by the Division of Chemical Sciences, Geosciences and Biosciences, Office of Basic Energy Sciences of Department of Energy grant DE-FG02-91ER20052 to F.D., by ISF 1466/18, BSF 2016070, and Ministry of Science and Technology 80802 grants to D.S.-D. Y. O. was supported by the grant GRK2202-23577276/RTG from DFG, Germany. Data analysis was partly supported by the NIH grant S10OD023592. This research was supported in part by the NCI, National Cryo-EM Facility at the Frederick National Laboratory for Cancer Research under contract HSSN261200800001E. The authors thank Ulrich Baxa, Thomas Edwards, and Adam Wier for their support and helpful discussions. Some cryo-EM data were also obtained at the University of Massachusetts Cryo-EM Core Facility, and we thank Dr. Chen Xu, Dr. KangKang Song, and Dr. Kyounghwan Lee for their support. Cryo-EM sample screening and optimization was performed at the Electron Microscopy Resource Laboratory at the Perelman School of Medicine, University of Pennsylvania, and we thank Dr. Sudheer Molugu for his support. We also thank Dr. S. Saif Hasan, Dr. Brian G. Pierce, and Dr. Christian Presley at the Institute for Bioscience and Biotechnology Research, University of Maryland for insightful discussions and invaluable help they provided during this study. S.S. and F.D. thank Vivian Kitainda for her assistance with protein purification and O2 consumption measurements.
© 2021, The Author(s).